Browsing by Subject "cholesterol"

Now showing 1 - 10 of 11
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    AFFINITY OF CHOLESTEROL FOR POLYUNSATURATED FATTY ACID-CONTAINING PHOSPHOLIPIDS
    (Office of the Vice Chancellor for Research, 2012-04-13) Kagimbi, Maureen W.; Williams, Justin A.; Wassall, Stephen R.
    A wide range of health benefits is associated with the consumption of omega-3 polyunsaturated fatty acids (PUFAs). One possible mechanism is that through our diet, they are incorporated into the phospholipids of the plasma membrane and disrupt the molecular organization of membrane do-mains due to the high disorder of PUFA. Our focus is the interaction of PUFA with cholesterol, a major component in plasma membranes. The objective here is to measure the affinity of cholesterol for PUFA-containing phospholip-ids by observing how cholesterol partitions between large unilamellar vesi-cles (LUVs) and Cyclodextrin (CD). Crucial to this determination, we need to be able to determine the concentration of cholesterol in LUVs and CD using an enzymatic colorimetric assay to create a standard curve of light absorb-ance (at 570nm wavelength) as a function of cholesterol concentration. The assay and its application to measuring binding coefficients for cholesterol will be described.
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    All n-3 PUFA are not the same: MD simulations reveal differences in membrane organization for EPA, DHA and DPA
    (Elsevier, 2018) Leng, Xiaoling; Kinnun, Jacob J.; Cavazos, Andres T.; Canner, Samuel W.; Shaikh, Saame Raza; Feller, Scott E.; Wassall, Stephen R.; Physics, School of Science
    Eicosapentaenoic (EPA, 20:5), docosahexaenoic (DHA, 22:6) and docosapentaenoic (DPA, 22:5) acids are omega-3 polyunsaturated fatty acids (n-3 PUFA) obtained from dietary consumption of fish oils that potentially alleviate the symptoms of a range of chronic diseases. We focus here on the plasma membrane as a site of action and investigate how they affect molecular organization when taken up into a phospholipid. All atom MD simulations were performed to compare 1-stearoyl-2-eicosapentaenoylphosphatylcholine (EPA-PC, 18:0–20:5PC), 1-stearoyl-2-docosahexaenoylphosphatylcholine (DHA-PC, 18:0–22:6PC), 1-stearoyl-2-docosapentaenoylphosphatylcholine (DPA-PC, 18:0–22:5PC) and, as a monounsaturated control, 1-stearoyl-2-oleoylphosphatidylcholine (OA-PC, 18:0–18:1PC) bilayers. They were run in the absence and presence of 20 mol% cholesterol. Multiple double bonds confer high disorder on all three n-3 PUFA. The different number of double bonds and chain length for each n-3 PUFA moderates the reduction in membrane order exerted (compared to OA-PC, 𝑆̅𝐶𝐷 = 0.152). EPA-PC (𝑆̅𝐶𝐷 = 0.131) is most disordered, while DPA-PC ( 𝑆̅𝐶𝐷 = 0.140) is least disordered. DHA-PC (𝑆̅𝐶𝐷 = 0.139) is, within uncertainty, the same as DPA-PC. Following the addition of cholesterol, order in EPA-PC (𝑆̅𝐶𝐷 = 0.169), DHA-PC (𝑆̅𝐶𝐷 = 0.178) and DPA-PC (𝑆̅𝐶𝐷 = 0.182) is increased less than in OA-PC (𝑆̅𝐶𝐷 = 0.214). The high disorder of n-3 PUFA is responsible, preventing the n-3 PUFA-containing phospholipids from packing as close to the rigid sterol as the monounsaturated control. Our findings establish that EPA, DHA and DPA are not equivalent in their interactions within membranes, which possibly contributes to differences in clinical efficacy.
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    Cholesterol Sulfonation Enzyme, SULT2B1b, Modulates AR and Cell Growth Properties in Prostate Cancer
    (AACR Publications, 2016-09) Vickman, Renee E.; Crist, Scott A.; Kerian, Kevin; Eberlin, Livia; Cooks, R. Graham; Burcham, Grant N.; Buhman, Kimberly K.; Hu, Chang-Deng; Mesecar, Andrew D.; Cheng, Liang; Ratliff, Timothy L.; Pathology and Laboratory Medicine, School of Medicine
    Cholesterol accumulates in prostate lesions and has been linked to prostate cancer (PCa) incidence and progression. However, how accumulated cholesterol contributes to PCa development and progression is not completely understood. Cholesterol sulfate (CS), the primary sulfonation product of cholesterol sulfotransferase (SULT2B1b), accumulates in human prostate adenocarcinoma and precancerous prostatic intraepithelial neoplasia (PIN) lesions compared to normal regions of the same tissue sample. Given the enhanced accumulation of CS in these lesions, it was hypothesized that SULT2B1b-mediated production of CS provides a growth advantage to these cells. To address this, PCa cells with RNAi-mediated knockdown (KD) of SULT2B1b were used to assess the impact on cell growth and survival. SULT2B1b is expressed and functional in a variety of prostate cells and the data demonstrate that SULT2B1b KD, in LNCaP and other androgen-responsive (VCaP and C4-2) cells, results in decreased cell growth/viability and induces cell death. SULT2B1b KD also decreases androgen receptor (AR) activity and expression at mRNA and protein levels. While AR overexpression has no impact on SULT2B1b KD-mediated cell death, addition of exogenous androgen is able to partially rescue the growth inhibition induced by SULT2B1b KD in LNCaP cells. These results suggest that SULT2B1b positively regulates the AR either through alterations in ligand availability or by interaction with critical co-regulators that influence AR activity.
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    Cholesterol-Induced Buckling in Physisorbed Polymer-Tethered Lipid Monolayers
    (MDPI, 2013-04-06) Hussain, Noor F.; Siegel, Amanda P.; Johnson, Merrell A.; Naumann, Christoph A.; Chemistry and Chemical Biology, School of Science
    The influence of cholesterol concentration on the formation of buckling structures is studied in a physisorbed polymer-tethered lipid monolayer system using epifluorescence microscopy (EPI) and atomic force microscopy (AFM). The monolayer system, built using the Langmuir-Blodgett (LB) technique, consists of 3 mol % poly(ethylene glycol) (PEG) lipopolymers and various concentrations of the phospholipid, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and cholesterol (CHOL). In the absence of CHOL, AFM micrographs show only occasional buckling structures, which is caused by the presence of the lipopolymers in the monolayer. In contrast, a gradual increase of CHOL concentration in the range of 0–40 mol % leads to fascinating film stress relaxation phenomena in the form of enhanced membrane buckling. Buckling structures are moderately deficient in CHOL, but do not cause any notable phospholipid-lipopolymer phase separation. Our experiments demonstrate that membrane buckling in physisorbed polymer-tethered membranes can be controlled through CHOL-mediated adjustment of membrane elastic properties. They further show that CHOL may have a notable impact on molecular confinement in the presence of crowding agents, such as lipopolymers. Our results are significant, because they offer an intriguing prospective on the role of CHOL on the material properties in complex membrane architecture.
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    Classic and targeted anti-leukaemic agents interfere with the cholesterol biogenesis metagene in acute myeloid leukaemia: Therapeutic implications
    (Wiley, 2020-05-25) Chen, Fangli; Wu, Xue; Niculite, Cristina; Gilca, Marilena; Petrusca, Daniela; Rogozea, Adriana; Rice, Susan; Guo, Bin; Griffin, Shawn; Calin, George A.; Boswell, H. Scott; Konig, Heiko; Medicine, School of Medicine
    Despite significant advances in deciphering the molecular landscape of acute myeloid leukaemia (AML), therapeutic outcomes of this haematological malignancy have only modestly improved over the past decades. Drug resistance and disease recurrence almost invariably occur, highlighting the need for a deeper understanding of these processes. While low O2 compartments, such as bone marrow (BM) niches, are well‐recognized hosts of drug‐resistant leukaemic cells, standard in vitro studies are routinely performed under supra‐physiologic (21% O2, ambient air) conditions, which limits clinical translatability. We hereby identify molecular pathways enriched in AML cells that survive acute challenges with classic or targeted therapeutic agents. Experiments took into account variations in O2 tension encountered by leukaemic cells in clinical settings. Integrated RNA and protein profiles revealed that lipid biosynthesis, and particularly the cholesterol biogenesis branch, is a particularly therapy‐induced vulnerability in AML cells under low O2 states. We also demonstrate that the impact of the cytotoxic agent cytarabine is selectively enhanced by a high‐potency statin. The cholesterol biosynthesis programme is amenable to additional translational opportunities within the expanding AML therapeutic landscape. Our findings support the further investigation of higher‐potency statin (eg rosuvastatin)–based combination therapies to enhance targeting residual AML cells that reside in low O2 environments.
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    Excess membrane cholesterol is an early contributing reversible aspect of skeletal muscle insulin resistance in C57BL/6NJ mice fed a Western-style high-fat diet
    (American Physiological Society, 2019-08-06) Grice, Brian A.; Barton, Kelly J.; Covert, Jacob D.; Kreilach, Alec M.; Tackett, Lixuan; Brozinick, Joseph T.; Elmendorf, Jeffrey S.; Anatomy and Cell Biology, School of Medicine
    Skeletal muscle insulin resistance manifests shortly after high-fat feeding, yet mechanisms are not known. Here we set out to determine whether excess skeletal muscle membrane cholesterol and cytoskeletal derangement known to compromise glucose transporter (GLUT)4 regulation occurs early after high-fat feeding. We fed 6-wk-old male C57BL/6NJ mice either a low-fat (LF, 10% kcal) or a high-fat (HF, 45% kcal) diet for 1 wk. This HF feeding challenge was associated with an increase, albeit slight, in body mass, glucose intolerance, and hyperinsulinemia. Liver analyses did not reveal signs of hepatic insulin resistance; however, skeletal muscle immunoblots of triad-enriched regions containing transverse tubule membrane showed a marked loss of stimulated GLUT4 recruitment. An increase in cholesterol was also found in these fractions from HF-fed mice. These derangements were associated with a marked loss of cortical filamentous actin (F-actin) that is essential for GLUT4 regulation and known to be compromised by increases in membrane cholesterol. Both the withdrawal of the HF diet and two subcutaneous injections of the cholesterol-lowering agent methyl-β-cyclodextrin at 3 and 6 days during the 1-wk HF feeding intervention completely mitigated cholesterol accumulation, cortical F-actin loss, and GLUT4 dysregulation. Moreover, these beneficial membrane/cytoskeletal changes occurred concomitant with a full restoration of metabolic responses. These results identify skeletal muscle membrane cholesterol accumulation as an early, reversible, feature of insulin resistance and suggest cortical F-actin loss as an early derangement of skeletal muscle insulin resistance.
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    Exercise training prevents skeletal muscle plasma membrane cholesterol accumulation, cortical actin filament loss, and insulin resistance in C57BL/6J mice fed a western‐style high‐fat diet
    (Wiley, 2017-08-15) Ambery, Ashley G.; Tackett, Lixuan; Penque, Brent A.; Brozinick, Joseph T.; Elmendorf, Jeffrey S.; Cellular and Integrative Physiology, School of Medicine
    Insulin action and glucose disposal are enhanced by exercise, yet the mechanisms involved remain imperfectly understood. While the causes of skeletal muscle insulin resistance also remain poorly understood, new evidence suggest excess plasma membrane (PM) cholesterol may contribute by damaging the cortical filamentous actin (F‐actin) structure essential for GLUT4 glucose transporter redistribution to the PM upon insulin stimulation. Here, we investigated whether PM cholesterol toxicity was mitigated by exercise. Male C57BL/6J mice were placed on low‐fat (LF, 10% kCal) or high‐fat (HF, 45% kCal) diets for a total of 8 weeks. During the last 3 weeks of this LF/HF diet intervention, all mice were familiarized with a treadmill for 1 week and then either sham‐exercised (0 m/min, 10% grade, 50 min) or exercised (13.5 m/min, 10% grade, 50 min) daily for 2 weeks. HF‐feeding induced a significant gain in body mass by 3 weeks. Sham or chronic exercise did not affect food consumption, water intake, or body mass gain. Prior to sham and chronic exercise, “pre‐intervention” glucose tolerance tests were performed on all animals and demonstrated that HF‐fed mice were glucose intolerant. While sham exercise did not affect glucose tolerance in the LF or HF mice, exercised mice showed an improvement in glucose tolerance. Muscle from sham‐exercised HF‐fed mice showed a significant increase in PM cholesterol, loss of cortical F‐actin, and decrease in insulin‐stimulated glucose transport compared to sham‐exercised LF‐fed mice. These HF‐fed skeletal muscle membrane/cytoskeletal abnormalities and insulin resistance were improved in exercised mice. These data reveal a new therapeutic aspect of exercise being regulation of skeletal muscle PM cholesterol homeostasis. Further studies on this mechanism of insulin resistance and the benefits of exercise on its prevention are needed.
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    FoxO3 transcription factor and Sirt6 deacetylase regulate LDL-cholesterol homeostasis via control of the proprotein convertase subtilisin/kexin type 9 (Pcsk9) gene expression
    (2013-08) Tao, Rongya; Xiong, Xiwen; DePinho, Ronald A.; Deng, Chu-Xia; Dong, X. Charlie; Department of Biochemistry and Molecular Biology, IU School of Medicine
    Elevated LDL-cholesterol is a risk factor for the development of cardiovascular disease. Thus, proper control of LDL-cholesterol homeostasis is critical for organismal health. Genetic analysis has identified PCSK9 (proprotein convertase subtilisin/kexin type 9) as a crucial gene in the regulation of LDL-cholesterol via control of LDL receptor degradation. Although biochemical characteristics and clinical implications of PCSK9 have been extensively investigated, epigenetic regulation of this gene is largely unknown. In this work we have discovered that Sirt6, an NAD+-dependent histone deacetylase, plays a critical role in the regulation of the Pcsk9 gene expression in mice. Hepatic Sirt6 deficiency leads to elevated Pcsk9 gene expression and LDL-cholesterol as well. Mechanistically, we have demonstrated that Sirt6 can be recruited by forkhead transcription factor FoxO3 to the proximal promoter region of the Pcsk9 gene and deacetylates histone H3 at lysines 9 and 56, thereby suppressing the gene expression. Also remarkably, overexpression of Sirt6 in high fat diet-fed mice lowers LDL-cholesterol. Overall, our data suggest that FoxO3 and Sirt6, two longevity genes, can reduce LDL-cholesterol levels through regulation of the Pcsk9 gene.
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    Lipid bilayer thickness determines cholesterol's location in model membranes
    (RSC, 2016-12) Marquardt, Drew; Heberle, Frederick A.; Greathouse, Denise V.; Koeppe, Roger E., II.; Standaert, Robert F.; Van Oosten, Brad J.; Harroun, Thad A.; Kinnun, Jacob J.; Williams, Justin A.; Wassall, Stephen R.; Katsaras, John; Department of Physics, School of Science
    Cholesterol is an essential biomolecule of animal cell membranes, and an important precursor for the biosynthesis of certain hormones and vitamins. It is also thought to play a key role in cell signaling processes associated with functional plasma membrane microdomains (domains enriched in cholesterol), commonly referred to as rafts. In all of these diverse biological phenomena, the transverse location of cholesterol in the membrane is almost certainly an important structural feature. Using a combination of neutron scattering and solid-state 2H NMR, we have determined the location and orientation of cholesterol in phosphatidylcholine (PC) model membranes having fatty acids of different lengths and degrees of unsaturation. The data establish that cholesterol reorients rapidly about the bilayer normal in all the membranes studied, but is tilted and forced to span the bilayer midplane in the very thin bilayers. The possibility that cholesterol lies flat in the middle of bilayers, including those made from PC lipids containing polyunsaturated fatty acids (PUFAs), is ruled out. These results support the notion that hydrophobic thickness is the primary determinant of cholesterol's location in membranes.
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    PCSK9 Inhibitors for LDL Lowering
    (Elsevier, 2015-10) Walsh, James P.; Department of Medicine, IU School of Medicine